Together with oils, fats and proteins, polysaccharides such as starch are the essential renewable raw materials from plants.
A decisive obstacle to the use of renewable raw materials as industrial raw materials is the lack of materials whose form, structure or other physicochemical parameters precisely meet the requirements of the chemical industry. Two particular requirements of a raw material suitable for industrial use are that it is available in high purity and that it has a uniform chemical structure. The latter is important for ensuring that reactions proceed homogeneously during processing.
Although starch is a polymer made up of chemically uniform basic structural units, namely glucose molecules, it is a complex mixture of very varied molecular forms which differ in their degree of polymerization and the occurrence of branches in the glucose chains. The degree of branching determines inter alia the physicochemical properties of the starch in question and hence also its suitability for a very wide variety of applications. A distinction is made in particular between amylose starch, which is an essentially unbranched polymer made up of .alpha.-1,4-linked glucose molecules, and amylopectin starch, which in turn is a complex mixture of variously branched glucose chains. The branches arise from the occurrence of additional .alpha.-1,6 linkages.
In typical plants for starch production, such as maize or potato for example, the two forms of starch occur in proportions of about 25 parts of amylose to 75 parts of amylopectin.
To adapt the starch raw material to the different industrial applications, i.e. to vary its physicochemical properties, it is necessary inter alia to be able to influence the degree of branching of the starch.
With regard to the suitability of a basic material such as starch for its use in the industrial sector, it therefore seems desirable to provide processes for the production of amylogenic plants synthesizing a starch which is modified in comparison with the naturally occurring starch.
It is especially desirable to modify starch so that it has a modified degree of branching, e.g. a decrease or increase in the degree of branching, thereby forming a more uniform starch with a higher or lower amylose content.
An example of another property of interest for the industrial use of starch is the content of phosphate groups. Phosphate-containing starch has a broad application in a very wide variety of fields, e.g. in paper manufacture, in textile manufacture, as adhesives, in the food sector or in medicine. Furthermore, starch phosphate derivatives are suitable for use as emulsifiers. As the starch which occurs naturally in the majority of amylogenic plants contains only a very small proportion of phosphate groups, a specific exception here being starch formed in underground organs such as e.g. roots or potato tubers, phosphate groups have hitherto usually been introduced by means of chemical processes. To avoid the additional outlay on costs and time associated with such processes for the introduction of phosphate groups into starch, it seems desirable to provide processes which make it possible to modify plants so that they produce a starch which is modified in such a way as to have an increased content of phosphate groups.
As regards the degree of branching of starch, it is already known that for certain plant species, for example maize, varieties containing only amylopectin can be produced by mutagenesis, in which individual genes of the plant are inactivated. Likewise, for potatoes, a genotype which forms no amylose has been produced by the chemical mutagenesis of a haploid line (Hovenkamp-Hermelink et al., 1987, Theor. Appl. Genet. 75, 217-221). However, haploid lines, or the homozygous diploid or tetraploid lines developed therefrom, are unusable in agriculture. The mutagenesis technique is not applicable to the heterozygous tetraploid lines of agricultural interest since the inactivation of all the copies of a gene is technically impossible because of the presence of four different copies of the genotype.
Maize and pea varieties capable of producing amylose starch are also known, but the amylose concentration in the starch of these plants is only 60-80%. Furthermore, the mutagenesis process on which the production of these varieties is based cannot be applied to other plants, e.g. potatoes.
Visser et al. (1991, Mol. Gen. Genet. 225, 289) have moreover disclosed that potato varieties which form substantially pure amylopectin starch can be produced with the aid of genetic engineering methods, especially by antisense inhibition of the gene for the starch synthase bound to the starch granule.
WO 92/14827 has disclosed a branching enzyme of potato. This enzyme is designated as the Q enzyme (branching enzyme) of Solanum tuberosum. It is further known that with the aid of DNA sequences which contain the information for the branching enzyme of potato described in WO 92/14827, it is possible to produce transgenic plants in which the amylose/amylopectin ratio of the starch is modified, although the plants described in WO 92/14827 do not form a starch with a high amylose content.
The synthesis, degradation and modification of starch involves a large number of enzymes whose interaction has so far been only partially explained.
In the potato, the starch is synthesized primarily by the action of the starch synthase, which utilizes essentially ADP-glucose as the substrate for transferring a glucose residue onto the non-reducing end of a polyglucan. What other enzymes are involved in the synthesis of branched starch in the potato is largely unknown at the present time.
Again, several enzymes are involved in the modification and degradation of starch:
The starch phosphorylases, which utilize inorganic phosphate as a cosubstrate, degrade .alpha.-1,4 linkages up to four units before an .alpha.-1,6 branch and work from the non-reducing end. As an exoamylase, .beta.-amylase has a high specificity for .alpha.-1,4 linkages. The least polymerized substrate is maltotetraose. Branching points bring an end to chain degradation, the last .alpha.-1,4 linkage before the branching point staying intact (Whelan, 1961, Nature 190, 954-957). The remaining polyglucans can be processed by transglycosylases, which possess both hydrolytic and synthesizing enzymic activity.
The Q enzyme (branching enzyme) and the T enzyme work as transglycosylases on the modification of the starch. The minimal substrate for the reaction catalyzed by the T enzyme is .alpha.-1,4-maltose, which is converted to the trisaccharide panose and glucose, forming an .alpha.-1,6 linkage (Whelan, 1961; Abdullah & Whelan, 1960, J. Biochem. 75, 12P). The Q enzyme catalyzes the same transglycosylation exclusively on glucans with a chain length of at least 40 units. Whereas the occurrence of several Q enzymes is known for other species such as maize (Singh & Preiss, 1985, Plant Physiol. 79, 34-40), only the branching enzyme described in WO 92/14827 has been detected in the case of Solanum tuberosum.
Another transglycosylase, the D enzyme, was first described in 1953 (in Nature 172, 158). Peat et al. (1956, J. Chem. Soc., Part XX, 44-55) describe it as a transglycosylase which transfers maltodextrin sub strates with two or more units, thereby producing exclusively .alpha.-1,4 linkages. The substrate must be made up of at least three units and there is a low specificity in respect of the acceptor. Takaha et al. (1993, J. Biol. Chem. 268, 1391-1396) describe the purification of the D enzyme of potato (EC 2.4.1.25) and the cloning of a cDNA. The purified enzyme accepts glucose as the recipient of the chain to be transferred, but the donor must be made up of at least three glucose units.
The authors do not describe what type of linkage is formed in the transglycosylation and assume that the D enzyme, or disproportionation enzyme, is probably involved not so much in the modification as in the degradation of the starch. It is not yet known whether other enzymes are involved in the modification of the starch. It is also not known what effects the modification of the activities of the T or D enzyme has on the structure of the starch formed in the cells, nor have said effects been studied to date. Since it has been supposed hitherto that the D enzyme is involved in the degradation of the starch, an inactivation or over-expression of the gene coding for the D enzyme would not be expected to have any effect on the basic structure of the starch formed.
Nothing is currently known about the enzymic reactions involved in the introduction of phosphate groups into starch. Enzymes responsible for the introduction of the phosphate groups have not been identified, nor is there a clear explanation as to which substrates act as phosphate group donors.
Therefore, no-one has yet succeeded in specifically modifying amylogenic plants by genetic engineering methods so that a starch can be produced therein which, in terms of its physicochemical properties, for example the amylose content or the degree of branching, is modified in such a way that it is more suitable for industrial processing than starch which occurs naturally in plants.
Furthermore, it is not yet known how an amylogenic plant can be specifically modified by means of genetic engineering methods so that the starch formed in these plants has a higher content of phosphate groups.